Study of NPS1, a novel CDC gene of cerevisiae

酿酒酵母CDC新基因NPS1的研究

• 批准号: 04660122
• 负责人: TSUCHIYA Eiko
• 金额: $ 1.41万
• 依托单位: HIROSHIMA UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1992
• 资助国家: 日本
• 起止时间: 1992 至 1993
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-04660122/
• 关键词: Saccharomyces cerevisiae; Cell-Division Cycle; G2-Phase Control; DNA-Binding Protein; Protein Kinase; Nuclear Protein; Saccharomyces cevevisiae; プロテインチナーゼ

The NPSl gene is a novel cell division cycle (CDC) gene of Saccharomyces cerevisiae enconding a 160 kd nuclear protein essenntial for cell growth. I investigated the functions of NPS1 gene in cell cycle and obtained the following results.1) I constructed a mutant nps1 gene which has a glutamic acid residue at position 792 instead of lysin residue (Lys792) at a suspected ATP-binding site of protein kinase consennsus motief, and found that Lys792 is indispensable for in vivo function of NPS1 gene product.2) By investigating the phenotypes of conditional lethal strain which can express NPS1 gene only in galactose medium, I found that nps1 gene works in g2 phase of S.cerevisiae transducing the signal of completion of DNA replication.3) The NPS1 gene product was found to locate in nuclear saffold of S.cerevisiae and possess DNA-binding activity.4) I succeeded to produce the Nps1 protein in SF9 cell transformed with recombinant baculovirus carrying NPS1 gene. Large scaled culture of transformed SF9 cells and purification of Nps1 protein are now on going.5) In order to identify genes acting in relation to NPS1, I carried out the following experiments : i) isolate mutant whose growth is suppressed by the high dosage-expression of NPS1 gene ; ii) screen the high copy suppressor of lethality caused by NPS1 depletion ; iii) screen the gene which cause synthetic lethality with high-dosage expression of NPS1 gene. The analyzes of mutants and genes obtained by the above experiments are now on going.

NPS1基因是酿酒酵母的新型细胞分裂周期(CDC)基因，编码细胞生长必需的160kd核蛋白。我研究了NPS1基因在细胞周期中的功能，得到了以下结果。1）我构建了一个突变的nps1基因，该基因在792位具有谷氨酸残基，而不是蛋白激酶共有基序的疑似ATP结合位点上的赖氨酸残基（Lys792），并发现Lys792对于NPS1基因产物的体内功能是不可缺少的。2） 在研究仅在半乳糖培养基中表达NPS1基因的条件致死菌株的表型时，我发现nps1基因在酿酒酵母的g2期起作用，转导DNA复制完成的信号。3）NPS1基因产物被发现位于酿酒酵母的核支架中，并具有DNA结合活性。4）我成功地在SF9细胞中产生了Nps1蛋白 用携带NPS1基因的重组杆状病毒转化。转化SF9细胞的大规模培养和Nps1蛋白的纯化正在进行中。5）为了鉴定与NPS1相关的基因，我进行了以下实验：i）分离出NPS1基因高剂量表达抑制其生长的突变体； ii) 筛选由 NPS1 缺失引起的高致死性复制抑制因子； iii)通过高剂量表达NPS1基因筛选引起合成致死的基因。对上述实验获得的突变体和基因的分析正在进行中。

项目成果

E.Tsuchiya: "The Saccharomyces cerevisiae NPSI gene,a novel CDC gene which encodes a 160 kDa nuclear protein involved in G2 phase control" EMBO Journal. 11. 4017-4026 (1992)

E.Tsuchiya：“酿酒酵母 NPSI 基因，一种新型 CDC 基因，编码参与 G2 期控制的 160 kDa 核蛋白”EMBO 杂志。

E.Tsuchiya and T.Miyakawa: "Nuclear structure and cell cycle control" Protein, Nucleic Acid and Enzyme. 39, (in press). (1994)

E.Tsuchiya 和 T.Miyakawa：“核结构和细胞周期控制”蛋白质、核酸和酶。

土屋英子: "細胞周期と核構造" 蛋白質 核酸 酵素. 39(印刷中). (1994)

Eiko Tsuchiya：“细胞周期和核结构”蛋白质核酸酶 39（出版中）。

E.Tsuchiya: "The Saccharomyces cerevisiae NPS1 gene,a novel CDC gene which encodes a 160 kDa nuclear protein involved in G2 phase control" EMBO Journal. 11. 4017-4026 (1992)

E.Tsuchiya：“酿酒酵母 NPS1 基因，一种新型 CDC 基因，编码参与 G2 期控制的 160 kDa 核蛋白”EMBO 杂志。

E.Tsuchiya et, al.: "The Saccharomyces cerevisiae NPS1 gene, a novel CDC gene which encodes a 160 kDa nuclear protein involved in G2 phase control" EMBO Journal. 11-11. 4017-4026 (1992)

E.Tsuchiya 等人：“酿酒酵母 NPS1 基因，一种新型 CDC 基因，编码参与 G2 期控制的 160 kDa 核蛋白”EMBO 杂志。